Various publications, which may include patents, published applications, technical articles and scholarly articles, are cited throughout the specification in parentheses, and full citations of each may be found at the end of the specification. Each of these cited publications is incorporated by reference herein, in its entirety.
Δ9-Tetrahydrocannabinol (THC) is the main psychoactive substance found in the cannabis plant. THC activates two distinct G protein-coupled receptors, cannabinoid 1 receptor (CB1) and cannabinoid 2 receptor (CB2) (Matsuda et al. 1990; Munro et al. 1993). CB1 is primarily expressed in the central nervous system (CNS) (Hohmann and Herkenham 1999; Farquhar-Smith et al. 2000; Rice et al. 2002; Walczak et al. 2005). CB2 expression, however, seems to be restricted to only peripheral tissues (Munro et al. 1993; Galiegue et al. 1995).
CNS mediated analgesic effects of cannabinoids have been well documented, but there is also accumulating evidence suggesting that cannabinoids can produce antinociception through peripheral mechanisms involving CB1 or CB2 (Hohmann 2002) (Malan et al. 2002). Richardson et al. demonstrated that cannabinoid antihyperalgesic effects were predominantly mediated by CB1 (Richardson et al. 1998; Richardson 2000). Hanus et al. showed that intraperitoneal injection of a CB2 selective agonist could suppress the late-phase response in the formalin test (Hanus et al. 1999). It was also shown that a CB2 selective agonist could attenuate thermal nociception and hyperalgesia (Malan et al. 2001; Malan et al. 2002; Quartilho et al. 2003) or suppress hyperalgesia evoked by intradermal administration of capsaicin (Hohmann et al. 2004). Ibrahim et al. showed that activation of CB2 with a selective CB2 agonist inhibited experimental neuropathic pain (Ibrahim et al. 2006). Taken together, the accumulating evidence clearly suggests great potential therapeutic value in targeting CB2 as a peripheral target for the treatment of pain. It should be noted that a significant advantage of this approach is that it would preclude unwanted CNS side effects caused by targeting CB1.
An arachidonic acid derivative, 2-arachidonyl glycerol (2-AG), is one of the two major and most well studied endogenous ligands for CB1 and CB2 (Gonsiorek et al. 2000). It has been shown that 2-AG acts as a potent and full-efficacy agonist of CB2 (Gonsiorek et al. 2000; Sugiura et al. 2000; Maresz et al. 2005) and that 2-AG is primarily hydrolysed by monoacylglycerol lipase (MGLL) (Dinh et al. 2002; Dinh et al. 2004; Saario et al. 2004). A non-competitive MGLL inhibitor that blocked 2-AG hydrolysis was found to enhance 2-AG levels and antinociception in stress models (Hohmann et al. 2005; Makara et al. 2005). It was also demonstrated that local administration of either 2-AG or a selective MGLL inhibitor induced a dose-dependent antinociceptive effect in an inflammatory pain model. Furthermore, local administration of the selective MGLL inhibitor in combination with 2-AG produced an additive antinociceptive effect (Guindon et al. 2007). Thus selective inhibition of MGLL may provide a novel therapeutic approach for the treatment of pain. Hitting this target, however, is inconceivable without good knowledge of the enzyme (Vandevoorde and Lambert 2005).
Lipases are lipolytic enzymes that have been differentiated from carboxylesterases by the fact that lipases have improved kinetics of hydrolysis for emulsions formed in oversaturated solutions. Carboxylesterases have been shown to have maximal activity using solutions of short-chain esters, with half-maximal activity at substrate concentrations far below the solubility limit. Exceeding the solubility limit was shown to have no effect on carboxylesterase activity. Lipases, on the other hand, were shown to have maximal activity using emulsified substrates, with half-maximal activity at substrate concentrations near the solubility limit (Chahinian et al. 2002). Early work with porcine pancreatic lipase showed that activity was low using a solution of ester substrates and abruptly increased as soon as an emulsion was formed. It was speculated that the porcine pancreatic lipase was activated by a conformational change of the enzyme as it bound to its water-insoluble substrate. The work with porcine pancreatic lipase was reviewed by Nini et al. (Nini et al. 2001).
In general, lipases share a similar α/β hydrolase fold with a catalytic Ser-His-Asp triad buried beneath a flexible cap-domain which is also referred to as a “lid” or “flap” (Brady et al. 1990; Winkler et al. 1990; Schrag et al. 1991). Although there is little conservation in the primary sequence of the cap-domain, it is generally formed of loops and one or more amphipatic helices. The cap-domains of human and dog gastric lipase are composed of intricate mixtures of 8 helices, turns, and random coils (Roussel et al. 1999; Roussel et al. 2002). In the crystal structure of human pancreatic lipase the lid adopts a helix-turn helix motif composed of two short amphipathic helices (van Tilbeurgh et al. 1992).
It has long been proposed that higher lipase activity for substrates presented as multimolecular aggregates (inter facial activation) is due to a conformational change in the cap-domain. It has also been proposed that changes from a closed to an open conformation of the lid is triggered by interaction with the substrate or lipid membrane (Brzozowski et al. 1991; van den Berg et al. 1995; van den Berg et al. 1995; Nini et al. 2001). Several other reports have also indicated that the loop covering the active site mediates lipase substrate specificity (Dugi et al. 1992; Dugi et al. 1995). It was demonstrated that movement of the helical lid results in a change in the hydrophobic-hydrophilic balance of the exterior surface of the lipase with the hydrophobic side of the lid becoming completely exposed in the active enzyme (Faustinella et al. 1992). Some lipases, such as guinea pig pancreatic lipase and bile salt-activated lipase, do not have a lid domain. Their active sites are freely accessible to solvent. As expected, based on the lack of the a cap-domain, these lipases are not activated by a lipid/water interface (Wang et al. 1997) (Carriere et al. 1997).
Although much has been learned about the structure of lipases through determination of three-dimensional structures of several microbial lipases and mammalian lipases, the three-dimensional structure of MGLL is unknown and its mechanism of action is not well understood. Furthermore, MGLL shows very little sequence similarity with other mammalian lipases and is unique among lipases in having monoglycerides as its only substrates. MGLL seems to be only distantly related to microbial proteins that include esterases, lysophospholipases, and haloperoxidases (Karlsson et al. 1997). A virtual molecular model of MGLL was built based on the crystal structure of chloroperoxidase (Saario et al. 2005; Saario et al. 2006). The model shows an alpha beta hydrolase fold with a lid domain comprised of four helices. The model, however, is only a virtual model and gives little insight into the actual mechanism of action of MGLL.
A crystal structure of MGLL would greatly facilitate the effort to discover MGLL selective inhibitors. A potential problem for crystallization experiments with MGLL is that detergents have been required to purify and stabilize MGLL in solution for both recombinant MGLL and MGLL from endogenous sources. (Tornqvist and Belfrage 1976; Somma-Delpero et al. 1995; Karlsson et al. 2000). Without detergent the purified MGLL protein was prone to aggregation. Crystallizing a detergent-solubilized protein into a structure of sufficient regularity to enable high-resolution X-ray crystallography can be problematic because well-ordered protein crystals can be difficult to obtain (U.S. Pat. No. 6,172,262B1).
The present invention provides a number of soluble engineered forms of monoacylglycerol lipase protein (MGLL) that do not require detergent for purification. The forms of MGLL provided by the present invention permit the expression and purification of protein suitable for identifying active agents in high-throughput screening and for crystallography. The present invention also provides a crystallized form of MGLL and descriptions of the X-ray diffraction patterns. Selective point mutations of hydrophobic residues in the cap-domain of MGLL generated soluble protein that did not require detergent for purification and stability. The protein displayed monomeric behaviour by gel filtration and was suitable for crystallization and high-throughput screening. In addition, selective mutation of surface lysine residues produced protein that generated crystals of improved quality. The crystal structure of MGLL was determined at atomic resolution. The forms of MGLL provide protein that can be used to identify inhibitors in high-throughput screens and the crystal structure of MGLL provides an important tool for structure-based drug design of MGLL inhibitors.